This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Breast cancer malignancy is characterized by the spreading of cancerous cells into the lymphatic system. Accurate assessment of lymph nodes is a critical component in determining disease progression and subsequent therapeutic options available to patients. Currently, the most common practice of assessing lymph nodes is lymphadecnectomy, a microscopical examination of surgically removed lymph nodes. However, this painful and invasive procedure, which causes nodal destruction and swelling, is not always analytically reliable. Molecular imaging affords non-invasive options for visualizing lymph nodes. However, imaging techniques cannot accurately ascertain the malignancy of the infected lymph nodes, because they display only structures without revealing the bioactivities associated with disease progressions. We seek to develop imaging probes that highlight diseased lymph nodes by detecting a known breast cancer's biological indicator, Cathepsin-B. Cathepsin-B enzymatically cleaves proteins at specific amino acid sequences, and this activity is implicated in the invasion of cancerous cells into they lymph nodes by breaking down the basement membranes. Our proposed molecular probe utilizes Cathepsin-B's native cleaving activity as a specific activation mechanism. The inactive probe is a non-fluorescent compound composed of fluorescent dyes and quenchers attached onto a customized sequence of amino acids. It is activated to the fluorescent species by Cathepsin-B, which selectively cuts off the quenchers. This 'light switch'design reveals specific bioactivity information, which paves the way for non-invasive analytical imaging of lymph nodes. UCSF Mass Spectrometry Facility has the instrumentation and expertise for the characterization of our proposed probes. Mass spectrometry offers efficiency and ease of interpretation that is superior to other spectroscopic tools for our applications.